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Abstract

Objective—The postural tachycardia syndrome (POTS) has multiple symptoms, chief among which are tachycardia, weakness, and recurrent blackouts while standing. Previous research has implicated dysfunction of the norepinephrine transporter. A coding mutation in the norepinephrine transporter gene (SLC6A2) sequence has been reported in 1 family kindred only. The goal of the present study was to further characterize the role and regulation of the SLC6A2 gene in POTS.

Methods and Results—Sympathetic nervous system responses to head-up tilt were examined by combining norepinephrine plasma kinetics measurements and muscle sympathetic nerve activity recordings in patients with POTS compared with that in controls. The SLC6A2 gene sequence was investigated in leukocytes from POTS patients and healthy controls using single nucleotide polymorphisms genotyping, bisulphite sequencing, and chromatin immunoprecipitation assays for histone modifications and binding of the transcriptional regulatory complex, methyl-CpG binding protein 2. The expression of norepinephrine transporter was lower in POTS patients compared with healthy volunteers. In the absence of altered SLC6A2 gene sequence or promoter methylation, this reduced expression was directly correlated with chromatin modifications.

Conclusion—We propose that chromatin-modifying events associated with SLC6A2 gene suppression may constitute a mechanism of POTS.

Introduction

The postural tachycardia syndrome (POTS) has multiple symptoms,1 chief among which are symptomatic tachycardia, weakness, and recurrent blackouts while standing. Also common are impaired concentration and cognition while standing, postural presyncope, chest pain, tremor, headache, feverishness, anxiety, sleep disturbance, and vasospasm in the limbs, including Raynaud phenomenon. There is typically minimal fall in blood pressure on standing, despite the tendency to faint. The severity and course of the POTS is variable. In some patients the symptoms are rather mild but in others the disorder is so disabling as to render them effectively bedridden because of weakness and an inability to stand without fainting.

The causes of POTS remain uncertain and are probably multiple. Evidence exists to support the importance of excessive pooling of blood in the veins of the legs on standing2 and an oversensitive cardiopulmonary volume receptor response to venous pooling.2,3 Work by us,4 and others,5,6 but not all,7 also implicates deficiencies in the function or expression of the norepinephrine transporter (NET). In 1 family kindred, the cause of this has been traced to a point mutation in the coding region of the NET gene (SLC6A2) resulting in the production of a dysfunctional transporter protein.6 This defect, as expected, was found to augment the sympathoneural signal, most evidently in the heart, and to increase the rate of overflow of norepinephrine into plasma.6 In the remainder of POTS patients, no similar loss of function mutation has been identified, and the cause of the phenotype of impaired NET activity remains unknown.

Here, we have performed an extensive analysis of single nucleotide polymorphisms (SNPs) in the SLC6A2 gene in POTS patients and healthy volunteers, in addition to testing for an alternative epigenetic mechanism associated with impaired NET function. Although nominal association was found between 1 nonsynonymous SNP and the diagnosis of POTS, we found no genetic variant to which the POTS phenotype could be attributed. Without the ability to access the nuclei of sympathetic nerves in human subjects, we used leukocytes in our search for epigenetic factors, which may be influencing NET gene expression. Such approaches, using easily accessible peripheral tissues, have been successful in identifying a number of epigenetic markers in other diseases.8–12 Our results led us to the conclusion that irrespective of SLC6A2 gene sequence and CpG methylation, the chromatinization by methyl-CpG binding protein 2 (MeCP2) and histone modifications, which we document in the majority of POTS patients tested, is associated with the impairment of NET function phenotype in POTS and could be an important regulatory mechanism in the disorder.

Patients and Methods

Further details regarding the materials and procedures are provided in the online-only Data Supplement.

Recruitment

Patients with POTS were recruited through our Orthostatic Intolerance Clinic after exhaustive clinical evaluation to exclude any other relevant medical condition. All patients with POTS shared the common clinical characteristics of recurrent episodes of presyncope while standing, freedom from postural hypotension (fall in systolic blood pressure while standing in the clinic <20 mm Hg), but the presence of posture-related tachycardia (heart rate increase on standing recorded in the clinic >30 bpm). The epigenetic component of this study was performed using samples obtained from 22 unrelated patients with POTS (16 women, 6 men) and 40 healthy subjects (23 women, 17 men). Participant clinical data are presented in the Table.

Hemodynamics and Sympathetic Function While Supine in Control Subjects and Patients With POTS

Sympathetic Nerve Recording

To quantify the degree of sympathetic activity in POTS and healthy subjects, multiunit sympathetic nerve activity in postganglionic fibers distributed to the skeletal muscle vasculature was recorded by microneurography.13 Recording from the common peroneal nerve was performed in 8 patients with POTS and 8 healthy subjects.

Genetic Analyses of the SLC6A2 Gene

To account for the possible influence of polymorphisms in the SLC6A2 gene, including the single nucleotide polymorphism (SNP) reported by Shannon et al,6 the promoter region and exon 9 of the SLC6A2 gene were amplified with the use of the polymerase chain reaction and sense and antisense primers. Using the Sequenom MassARRAY platform (Sequenom, Inc., San Diego, CA) as previously described,14 93 other polymorphisms in the SLC6A2 gene, including synonymous and nonsynonymous SNPs, were also analyzed in DNA samples from a total of 65 healthy volunteers and 20 POTS patients.

Results

Hemodynamics and Sympathetic Function

Age, supine blood pressure, and body mass index were similar between the 2 groups, whereas resting heart rate was significantly elevated in the POTS patients (Table). Although supine heart rate was significantly elevated, muscle sympathetic nerve activity, expressed in terms of both burst frequency and burst incidence, was reduced in POTS patients. The ratio of norepinephrine spillover to plasma to multiunit sympathetic nerve activity burst frequency was substantially elevated in the POTS group, in keeping with impaired norepinephrine reuptake.

SLC6A2 Polymorphisms in POTS

Of the 16 naturally occurring polymorphisms (including the SNP causing the A457P mutation) known to encode a change in the amino acid sequence of the NET protein,15 which were tested, none were present in the 20 POTS patients analyzed (Table V in the online-only Data Supplement). One synonymous SNP, rs5564, was significantly associated with the diagnosis of POTS after adjustment for multiple testing (P=0.0014, target-α 0.0024). The rs5564 polymorphism lies within the splice site consensus sequence of exon 5. Because only a small number of patients were examined, this association requires validation in a larger cohort, and the possible functional significance also requires investigation.

SLC6A2 Promoter Methylation Is Unremarkable in POTS

The methylation of cytosine in the CpG dinucleotide distinguishes the epigenome, and this covalent modification is inversely associated with transcriptional expression.16 CpG sites occur in clusters called CpG islands, which are most commonly, but not exclusively, located in the promoters of genes.17 The human SLC6A2 gene has a CpG Island within the proximal promoter (Figure 1A). Region 1 was hypermethylated in both healthy and POTS individuals (81%–90% methylation of individual clones sequenced), whereas region 2 of the CpG Island was hypomethylated (9%–19% methylation), and region 3 was unmethylated (<1.5% methylation, Figure 1B). The data in region 3 are consistent with the unmethylated status of the CpG island proximal to the SLC6A2 gene transcription initiation start site in mouse neuronal cortical cells.18SLC6A2 genomic methylation was similar between the POTS patients and the healthy control subjects.

DNA methylation analysis of the human SLC6A2 gene in healthy and postural tachycardia syndrome (POTS) individuals. A, Schematic representation of the analysis of the 3 regions of the CpG Island within the proximal promoter of the SLC6A2 gene. B, Examples of the bisulphite genomic sequencing are shown for healthy and POTS individuals with multiple clones for the SLC6A2 gene. Specific methylated (black squares) and unmethylated (open squares) CpG dinucleotides are illustrated. The percentage CpG methylation determined by bisulphite sequencing is also accompanied in pie-graph. Regions 1 and 2 of SLC6A2 are predominantly hypermethylated and hypomethylated, respectively, whereas region 3 is largely unmethylated in healthy and POTS individuals. The arrow represents the transcriptional start site for SLC6A2.

Altered Gene Expression Patterns in POTS

Consistent with the above observations of unremarkable SLC6A2 gene methylation, we found no difference in the expression of the DNA methyltransferase DNMT1 in POTS patients (Figure 2A). Because the methylation of DNA is often associated with the binding of methyl-CpG determinants, we examined the methyl-CpG binding domain proteins, MBD1 and MeCP2. Although we found no difference in the expression of MBD1, (Figure 2B) we observed a small but significant change in MeCP2 expression in POTS patients (Figure 2C, P<0.005). Recent experimental evidence in the mouse cortical cells suggests SLC6A2 gene-activating complexes comprise regulatory determinants that mediate histone modifications.18 We, therefore, tested the expression of the H3K27m3 histone methyltransferase, EZH2 (Figure 2D), as well as the acetyltransferase p300. EZH2 mRNA levels were increased and inversely correlated with p300 in POTS individuals (Figure 2E; P<0.0001).

Histone Modifications Distinguish the SLC6A2 Promoter in POTS

A unique property of gene sequences is the polyvalent chromatin fiber, which is structurally and chemically altered to regulate gene expression. To address this, solubilized chromatin fractions were prepared in 6 healthy subjects and 6 POTS patients and immunopurified with antibodies that identify posttranslational modification of the histone H3 tail. We examined 3 regions of the SLC6A2 promoter sequence that included the CpG island. As shown in Figure 3A, the SLC6A2 gene is consistently deacetylated in POTS individuals for the gene-activating modification mark, histone H3 lysine 9/14 acetylation (H3K9/14ac, P<0.0001) in all 3 regions of the gene and demethylated in POTS for the gene-activating mark, histone H3 lysine 4 methylation (H3K4m3, P<0.001) in regions 1 and 2 (Figure 3B). These chromatin modifications were inversely correlated with the gene-suppressive modifications, histone H3 lysine 9 trimethylation (H3K9m3, Figure 3C) and lysine 27 trimethylation (H3K27m3) in all 3 regions of the gene in POTS patients (Figure 3D). This type of analysis also revealed similar results for the coregulatory determinant, MeCP2,19 which was clearly enriched on the promoter region of the SLC6A2 gene in POTS individuals when compared with healthy subjects (Figure 3E). The increased association of MeCP2 was tightly associated with H3K9m3 and H3K27m3 enrichment of the −872/−514 sequence of the SLC6A2 gene. To assess the specificity of MeCP2 chromatin binding, we also examined association of MeCP2 on nontarget genes, IFIH1 and FOXA1. The level of MeCP2 association on these genes did not significantly change between healthy and POTS patients (Figure II in the online-only Data Supplement). In the POTS patients H3K9m3, H3K27m3, and MeCP2 enrichment on the hNET gene were inversely correlated with the NET protein expression shown by Western blot (Figure 4). In comparison with healthy patients, we observed a significant reduction in NET protein expression in POTS relative to actin protein signal 0.66 (SD 0.043, SEM±0.031). Taken together, these results suggest that chromatin modifications may account for the impaired NET function. Protein detection was also validated using an alternate antibody to hNET (sab2102224, Sigma-Aldrich, Castle Hill, Australia) (Figure III in the online-only Data Supplement). NET mRNA expression was found to be extremely low in leukocytes; therefore quantitative comparison between patient groups was not performed.

Discussion

The present study identifies a change in NET expression in patients with POTS. This change in expression is attributable to increased binding of the repressive MeCP2 regulatory complex, in association with an altered histone modification composition at the promoter region of the SLC6A2 gene.

Possible dysfunction of the NET has previously been actively investigated in POTS patients, given the previous evidence in them for presence of faulty norepinephrine reuptake.4,6 Reuptake of norepinephrine into sympathetic nerves after its release terminates the neural signal. A fault in transmitter inactivation augments the effects of sympathetic nerve traffic. In the heart at least 80% of released noradrenaline is recaptured into sympathetic nerves,20 so the heart is more sensitive than the other organs to impairments in transmitter reuptake. Head-up tilt to 70° after selective pharmacological blockade of NET with reboxetine in healthy subjects generates a clinical phenotype that bears a close resemblance to that seen in POTS patients during tilt, with heart rate increasing by around 40 bpm.21 Our own data, documenting a reduction in expression of NET protein in sympathetic nerves accessed via subcutaneous vein biopsies in POTS patients, provides further support for deficiencies in NET as being important in POTS. To date, it has not been possible to study sympathetic nerve NET gene expression. The cell nuclei of sympathetic neurons are in sympathetic ganglia and cannot ethically be biopsied in POTS patients unlike the sympathetic fibers in the wall of subcutaneous veins.4 Contrary to these findings, elevated cardiac norepinephrine spillover to plasma in POTS patients in the absence of neurochemical evidence of an uptake defect has been previously described.7 In the present study, we estimated the proportionality of norepinephrine spillover to plasma per burst of sympathetic activity as an index of norepinephrine uptake, noting that the amount of norepinephrine overflowing into the circulation per sympathetic burst was significantly higher in the POTS patients, in keeping with impaired norepinephrine reuptake. Although this may have arisen from a defect in norepinephrine reuptake, it could also occur if the quanta of norepinephrine released per action potential was elevated. Further, if norepinephrine reuptake is reduced, this might be the consequence to swamping of the transporter by high synaptic levels of the transmitter in POTS. Supporting this premise, in response to yohimbine, cardiac norepinephrine spillover was elevated to a greater extent in POTS patients compared with controls,7 and once cardiac norepinephrine spillover exceeded ≈200 pmol/min, the cardiac extraction of tritiated norepinephrine declined appreciably, suggesting there was a diminution in the action of NET during periods of extreme sympathetic activation in POTS patients.

Direct sequence analysis of the SLC6A2 gene has been performed in many patients, but to date a loss of function coding region mutation of the SLC6A2 gene has been found in 1 family kindred only.6,22 Extensive analysis of SLC6A2 gene polymorphisms in the present study identified no genotype that could explain a dysfunction of NET in the majority of POTS patients. Given the evidence for the existence of the faulty neuronal norepinephrine reuptake phenotype in POTS patients, almost invariably in the absence of identifiable loss of function SLC6A2 gene coding region mutations, we tested for epigenetic modification of the SLC6A2 gene. Gene promoter region DNA methylation, involving the covalent addition of methyl groups at the 5' position of cytosine within CpG dinucleotides, is a major mechanism of gene regulation. Methylation occurring in CpG-rich islands of gene promoter regions is directly associated with the pathological silencing of the tumor suppressor genes in cancers, imprinting disorders, and gene-linked neurodevelopment syndromes.23 Silencing of transcription by promoter region DNA hypermethylation produces effects on phenotypes, which are usually indistinguishable from those caused by coding region mutations.24–26

The findings presented are novel at least in 2 aspects that identify a role of epigenetic regulation of NET in POTS patients. First, they demonstrate that in the POTS individuals, the promoter sequence of the SLC6A2 gene is subject to significant alterations of histone modifications that are associated with gene-suppressive transcriptional events. Chromatin immunopurifications and the detailed analysis of the 3 regions of the SLC6A2 gene indicate a substitution of gene-activating H3K9/14 acetylation and H3K4 trimethylation with gene-suppressive H3K9 and H3K27 trimethylation that is consistent with reduced NET expression in the POTS patients. Indeed, the importance of histone H3 tail modification of the SLC6A2 gene sequence is consistent with recent identification of epigenetic control in mouse neocortical cells.18 A major conclusion of these experiments in healthy individuals and POTS patients is the evidence that differential NET protein expression is associated with specific histone modifications.

Second, as genomic methylation was not associated with the binding of the MeCP2 repressor on SLC6A2, this result argues against methylation of the cytosine residue as the precedent determinant of silencing in POTS patients. Rather, experiments had identified H3K9 and H3K27 trimethylation to strongly correspond with MeCP2 association on the SLC6A2 gene. In this context, the unremarkable changes in CpG methylation observed between healthy individuals and POTS patients were less surprising. Recent findings are indicative of a multifunctional role for MeCP2 that is also found on nonmethylated CpG sequences. Indeed, our observations argue against the old model that has for so long relied on the paradigm of DNA methylation-mediated repression.18 The emerging picture for MeCP2 is remarkably complex with less exclusivity to methyl-CpG content and the possibility that its binding preferences are now broadened to unmethylated sequences.27 Distinct regulatory outcomes mediated by MeCP2 are also evident on unmethylated templates, and recent studies show, in its most simplest form, gene-activating epigenetic changes.28 In this context, our observations of unremarkable MeCP2 binding preferences for genomic methylation are unsurprising. Recent findings demonstrate that chromatinization by MeCP2 mediates silencing of unmethylated genes.29,30 The existence of distinct regulatory outcomes not exclusive to methyl-CpG content suggests a multifunctional role for MeCP2 with binding preferences that include histone modifications. A striking mechanistic commonality associated with MeCP2 cosuppression is the clear distinction of SLC6A2 gene silencing with histone H3K9m3 in cortical cells.18 If correct and analogous with reduced NET protein in POTS patients, the intricate H3K9m3 and H3K27m3 marks contextualizes the promoter in the course of histone deacetylation and reduced H3K4m3 to recognize MeCP2 to assist in downstream silencing (Figure 5). The changes we observed in mRNA expression require further experimental validation, and future studies could examine protein levels in the leukocytes of POTS. It is unclear at present why the expression of MECP2 and histone-modifying enzymes would be altered in the leukocytes of POTS patients and what further changes in gene expression at the global level may be occurring. Although this study has focused on changes at the NET gene, it may be worthwhile in the future to analyze the POTS transcriptome in detail using genome-wide sequencing.

Schematic representation of SLC6A2 gene expression in healthy individuals and postural tachycardia syndrome (POTS) patients. SLC6A2 gene activity is strongly associated with hyperacetylation (ac) of H3K9/14 and trimethylation of H3K4. These gene-activating histone modifications are inversely associated for the suppressive trimethylation (m3) mark on H3K9 and H3K27. Modification of SLC6A2 chromatin in POTS is associated with H3K9m3, H3K27m3, and histone deacetylation, as well as the recruitment of the methyl-CpG binding protein 2 (MeCP2) corepressor to assist in transcriptional suppression.

Studies by our group and others have shown that gene-regulating events involve transcription factors, remodeling enzymes, and chromatin modifications.18 Why the SLC6A2 gene in the leukocytes of POTS patients is suppressed is poorly understood. In noradrenergic cell types, the transcription factors Hand2 and Gata3 are regulated by cytokines and associated with SLC6A2 expression.31 Hand2 interacts directly with the histone acetyltransferase p300 to alter chromatin structure.31,32 We found reduced expression of p300 in POTS patients in this study. The dysregulation of transcriptional networks in POTS in relation to inflammation may be an important area for future research, given that POTS often develops after prolonged febrile illness.3

Why SLC6A2 chromatin in the leukocytes of POTS patients is modified with gene-suppressive histone marks remains unknown. Also unclear at present is whether therapeutic targeting of the SLC6A2 gene, or of the faulty neuronal norepinephrine reuptake phenotype, may prove to be of clinical value in the future. At the level of indirect pharmacological modification of the faulty reuptake phenotype, drugs available in the clinical arena can reduce norepinephrine reuptake, most notably the tricyclic antidepressants,20 but none have been demonstrated to augment reuptake. What relation impairment of norepinephrine reuptake in POTS patients has to their constellation of symptoms remains unknown, except perhaps for their tachycardia, which might be attributable to failure of clearance of norepinephrine from the cardiac sympathetic nerve synaptic spaces. For POTS patients, treatment in the foreseeable future will continue to involve exercise,33 agents such as the mineralocorticoid and fludrocortisone, which provides some protection against syncope by increasing plasma volume, and β-adrenergic blockade, for relief of symptomatic tachycardia.34 On the basis of the results presented here, however, in addition to our preclinical data35 further research into the potential application of histone-modifying drugs may hold promise

Sources of Funding

We acknowledge the grant and fellowship support from the National Health and Medical Research Council (NHMRC). This work was supported, in part, by the Victorian Government’s Operational Infrastructure Support Program.